P1354: CRISPR/CAS9 GENE EDITING OF IMMUNE CHECKPOINT RECEPTOR NKG2A IMPROVES THE ANTI-LEUKEMIC EFFICACY OF PRIMARY CD33-TARGETING CAR-NK CELLS.
Authors: Nawid Albinger, Tobias Bexte, Ahmad Al-Ajami, Philipp Wendel, Leon Buchinger, Alec Gessner, Jamal Alzubi, Vinzenz Särchen, Meike Vogler, Raj Bhayadia, Jan-Henning Klusmann, Nina Möker, Toni Cathomen, Michael A. Rieger, Katharina Imkeller, Evelyn Ullrich
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: CD33-targeting chimeric antigen receptor (CAR; CAR33)-T cells already showed efficacy for the treatment of acute myeloid leukemia (AML). Yet clinical application of CAR33-T cells remains challenging due to its restriction to autologous cell preparations and potential risk of severe side effects. In contrast, natural killer (NK) cells can be safely administered to HLA-mismatched recipients and possess an intrinsic killing capacity which can complement CAR-mediated cytotoxicity. Recently, we reported on the successful generation of primary CD33-targeting CAR33-NK cells, which are highly effective against AML in vitro as well as in AML-xenograft mouse models (Albinger et al., Blood Cancer J. 2022). Nevertheless, NK cell function can be impaired by high levels of the inhibitory immune checkpoint receptors such as NKG2A (natural killer group 2A) expressed on NK cells (Bexte et al., Oncoimmunology 2022). Aims: By applying CRISPR/Cas9 gene editing to knockout (KO) the killer cell lectin like receptor C1 (KLRC1) gene in CAR33-NK cells, we aimed to improved CAR33-NK cell functionality in vitro and in vivo. Additionally, using single cell sequencing technologies, we envisaged to analyse the effect of both CAR introduction and KO of KLRC1 on the RNA and protein expression in NK cells. Methods: CAR33-NK cells were generated by lentiviral transduction of peripheral blood-derived (PB-) NK cells. Nucleofection with CRISPR-Cas9 technology was used to KO the NKG2A-encoding KLRC1 locus in CAR33-NK cells. The CAR33- and NKG2A-expression as well as cytotoxicity were analysed using flow cytometry and IncuCyte® after feeder cell-free, IL-15/IL-2-based expansion. Changes in RNA and protein expression of gene-modified NK cells were determined using qPCR and cellular indexing of transcriptome and epitopes (CITE)-seq analysis. The in vivo-efficacy was evaluated in OCI-AML2 (GFP+, Luc+) xenografted NSG-SGM3 mouse models. Results: Lentiviral transduction of PB-NK cells resulted in up to 60% CAR33-positive cells, while KLRC1 gene disruption resulted in 50% reduction of NKG2A cell surface expression. CITE-Seq and qPCR analysis revealed a distinct gene regulation pattern in CAR33- and CAR33-KLRC1ko-NK cells. Upregulation of CXCR4, CD16 and CD70 in CAR33- and CAR33-KLRC1ko-NK cells indicated a mature and activated NK cell state. CAR33-KLRC1ko-NK cells showed significantly higher elimination of CD33+/HLA-E+ OCI-AML2 cells as well as primary patient material in in vitro cytotoxicity assays compared to KLRC1ko-NK or CAR33-NK cells. Furthermore, a reduction of leukemic burden was observed in vivo following a single injection of a low dose (3x106 cells) of CAR33-KLRC1ko-NK cells compared to CAR33-NK cell treatment in an NSG-SGM3 AML-xenograft mouse model. Two injections of 3x106 CAR33-KLRC1ko-NK cells each displayed superior efficacy compared to KLRC1ko-NK or CAR33-NK cell treatment and led to a complete elimination of AML and leukemia-initiating cells in the bone marrow, which was confirmed by bone marrow re-engraftment analysis. Application of CAR33-KLRC1ko-NK cells in vivo appeared to be safe and did not induce any side effects in line with histologic analysis of lung, liver and colon. Summary/Conclusion: Removing an inhibitory receptor in CAR-NK cells induced an anti-leukemic NK cell state which showed a highly beneficial effect for the treatment of AML. This double genetic modification has the potential to enable NK cells to bypass inhibition following contact with malignant cells not only in the context of AML, but also in a broad range of other malignant diseases. Keywords: NK cell, AML, Cellular therapy, NK receptor